Observation of peroxynitrite overproduction in cells during 5-fluorouracil treatment: Via a ratiometric fluorescent probe

Article abstract of DOI:10.1039/c9cc09652c

We describe a colorimetric and fluorescent probe 3a to detect cellular peroxynitrite with high selectivity and sensitivity. 3a was successfully applied in the bioimaging of exogenous and endogenous peroxynitrite in living cells. The up-regulation of peroxynitrite in cancer cells and normal cells during 5-fluorouracil treatment was finally monitored.

Full text of DOI:10.1039/c9cc09652c

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Observation of peroxynitrite overproduction
in cells during 5-fluorouracil treatment via
Cite this:DOI: 10.1039/c9cc09652c
a ratiometric fluorescent probe†
Received 12th December 2019,
Accepted 28th January 2020
ab
Ji-Ting Hou,
*
Bingya Wang,a Yongru Zhang,c Beibei Cui,a Xinhua Cao,a
Man Zhang,d Yong Ye *c and Shan Wang
*
a
DOI: 10.1039/c9cc09652c
rsc.li/chemcomm
We describe a colorimetric and fluorescent probe 3a to detect cellular
To date, ONOOÀ-specific fluorescent probes have been largely
peroxynitrite with high selectivity and sensitivity. 3a was successfully devised and used to monitor ONOOÀ fluctuations in various patho-
applied in the bioimaging of exogenous and endogenous peroxynitrite physiological processes,8–12 such as inflammation,8 drug-induced
in living cells. The up-regulation of peroxynitrite in cancer cells and organ toxicity,9 ischemia reperfusion injury,10 and diabetes.11 These
normal cells during 5-fluorouracil treatment was finally monitored.
reports have led to a better understanding of the roles of ONOOÀ in
certain biological courses. However, the development of ONOOÀ
Peroxynitrite (ONOOÀ) is a highly reactive oxygen speciesꢀ(ROS) bioimaging is still hindered by the availability of fluorescent probes
in living systems. It is formed by nitric oxide (NO) and O2 À in a with excellent optical properties and scarce pathophysiological
diffusion-controlled manner (k = 0.4–1.9 Â 1010 MÀ1
s
À1).1 models. For example, most of the reported probes for ONOOÀ
ONOOÀ plays key roles in maintaining the intracellular redox showed intensity changes at a single emission that are easily
balance due to its strong oxidability. In addition, it always interfered with by a variety of analyte-independent factors, such
functions as a signaling molecule and exhibits antimicrobial as instrumental performance, the intracellular microenvironment
and antibacterial activities.2 In contrast, aberrant ONOOÀ around the probe molecule, the heterogeneous distribution of the
expression in vivo is usually associated with cellular dysfunc- probe, and photobleaching. Thus, ratiometric fluorescent probes
tions because it damages a wide variety of biomacromolecules that show optical changes at two emission bands and can avoid
like DNA and proteins,3 resulting in diverse pathogenic effects, these interference sources for intensity-based probes are highly
such as neurodegenerative, cardiovascular and inflammatory desired for ONOOÀ measurement.13 In addition, some chemother-
diseases, and cancers.4 Therefore, it is important to develop apeutic agents can kill cancer cells by augmenting intracellular
reliable detection tools to monitor biological ONOOÀ. How- oxidative stress, like 5-fluorouracil (5-FU), which yet lacks sufficient
ever, efficient detection of intracellular ONOOÀ is challenging visual evidence.14 Therefore, new ratiometric fluorescent probes
because it exhibits an extremely short half-life (o10 ms) and are needed to monitor ONOOÀ fluctuation, to indirectly report
low steady-state concentration (nM range).5 Over the past oxidative stress during chemotherapy and for getting deeper
decade, fluorimetric analysis has been widely applied in the insights into the therapeutic mechanism of drugs.
bioimaging of ONOOÀ,6 because of its superb sensitivity, high
Here, we describe a series of phenothiazine-based fluorescent
temporal and spatial resolution, and non-invasive imaging probes 3a–3c. Phenothiazine was chosen as the fluorophore
ability.7
backbone because of its large Stokes shift and the reducibility
of the sulfur atom.15 The 2,3-diaminomaleonitrile was introduced
into the 3-position of phenothiazine to construct a CQN bond
that is susceptible to ROS.16 It is variable at the 2-position of
phenothiazine in these probes, and is supposed to affect their
optical behaviors under oxidative stress. We demonstrated that
2-position non-substituted probe 3a showed highly specific colori-
metric and fluorescent changes toward ONOOÀ in a ratiometric
manner, while 3b and 3c exhibited a similar response to ONOOÀ
and ClOÀ. Moreover, 3a was capable of detecting exogenous and
endogenous ONOOÀ in living cells. Finally, 3a was utilized to
visualize the generation of ONOOÀ in cancer cells and normal
cells treated by 5-FU, a drug used in the treatment of various
a College of Chemistry and Chemical Engineering, Xinyang Normal University,
Xinyang 464000, P. R. China. E-mail: houjiting2206@163.com, smallcoral@live.cn
b Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue,
College of Emergency and Trauma, Hainan Medical University, Haikou 571199,
China
c Phosphorus Chemical Engineering Research Center of Henan Province,
College of Chemistry, Zhengzhou University, Zhengzhou 450001, P. R. China.
E-mail: yeyong03@tsinghua.org.cn
d School of Chemistry and Materials Science, Hubei Engineering University,
Xiaogan 432000, P. R. China
† Electronic supplementary information (ESI) available: Synthesis methods and
supplementary figures. See DOI: 10.1039/c9cc09652c
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cancers.17 All of these probes were synthesized via a simple
procedure and characterized by 1H NMR, 13C NMR, and HRMS
(see ESI†).
We initially examined the optical response of probes 3a–3c to
reactive species, including ClOÀ, H2O2, ONOOÀ, O2 À, tBuOOH,
ꢀ
ꢀOH, S2À, and SO32À. In PBS solution (pH 7.2–7.4, 10 mM,
containing 1 mM Triton X-100), 3a, 3b, and 3c displayed two
main absorption bands at 435 nm and 347 nm, 454 nm and
343 nm, and 436 nm and 342 nm, respectively (Fig. S1 and S2,
ESI†). Upon the addition of the analytes, the absorption of 3b
and 3c obviously declined in the presence of ONOOÀ or ClOÀ,
while only ONOOÀ led to a loss of the absorption bands in 3a.
This also caused the generation of a new absorbance at 332 nm
with a color change from light yellow to colorless, indicating that
3a exhibited a higher selectivity toward ONOOÀ than the other
two probes. A similar phenomenon was also observed in fluores-
cence tests. 3b and 3c emitted at 640 nm and 605 nm, respec-
tively, while their fluorescence was largely quenched by ONOOÀ
and ClOÀ. To our delight, 3a showed an emission band at
630 nm, which blue-shifted to 480 nm upon the addition of
ONOOÀ with a 528-fold intensity ratio (I480/I630) enhancement
(Fig. 1). A slight ratio change was also found in the presence of
ClOÀ, which is negligible when compared to that induced by
ONOOÀ. Other reactive species caused no fluorescence changes
of 3a. Since multiple heteroatoms are comprised in the structure
of probe 3a, we further checked its fluorescence response toward
common metal ions, including Ca2+, Cu2+, Pb2+, Zn2+, and Ag+
(Fig. S3, ESI†). No obvious spectral variations were recorded,
demonstrating the high selectivity of 3a toward ONOOÀ. We note
here that the slight modification at the 2-position of phenothia-
zine leads to distinguishing optical behaviors of the probes
toward reactive species, which cannot be ignored for the design
of phenothiazine-based probes.
Fig. 2 Fluorescence titration of 3a toward various concentrations of
ONOOÀ in PBS solution (pH 7.2–7.4, 10 mM, containing 1 mM Triton
X-100). lex = 370 nm, slit width: 6 nm/6 nm. Inset: The photograph of 3a
before and after addition of ONOOÀ under 365 nm UV lamp.
ONOOÀ was calculated to be 19.4 nM signifying its high sensitivity.
The fluorescence titration experiment of 3a toward ClOÀ was also
performed to further verify its selectivity. Fig. S6 (ESI†) shows that
at 14 equiv. of ClOÀ, the intensity ratio change of 3a was limited,
again confirming the unique reaction between 3a and ONOOÀ.
Subsequently, the emission intensity ratio changes of 3a in
different pH buffered solutions were investigated (Fig. S7, ESI†).
The intensity ratio I480/I630 of 3a was unchanged from pH 3 to 10.
There was an obvious ratio enhancement upon the addition of
ONOOÀ in the same pH range implying the ability of 3a to
measure analytes over a wide pH range, including physiological
pH. In addition, the emission intensity of 3a with or without
ONOOÀ remained steady under continuous excitation for an
hour (Fig. S8, ESI†), confirming their excellent photostability,
which is an important feature for dynamic confocal bioimaging.
Time-dependent fluorescence emission changes in 3a (10 mM)
toward ONOOÀ (10 mM and 100 mM) were also exploited (Fig. S9,
ESI†). The emission intensity of 3a at 630 nm was sharply quenched
in seconds when various amounts of ONOOÀ were added, indicat-
ing a rapid reaction between the probe and ONOOÀ. Interestingly,
when 100 mM ONOOÀ was added, the fluorescence at 480 nm was
largely enhanced in the first few seconds, and then it gradually
increased and almost reached a plateau in 9 min. In contrast, when
10 mM ONOOÀ was added, the emission of 3a at 480 nm could
plateau in 1 min. This out-of-step change at two emission bands of
3a versus different amounts of ONOOÀ might be ascribed to the
multi-step oxidation of the probe by ONOOÀ.
The fluorescence response of 3a toward ONOOÀ was further
explored. The emission intensity of 3a at 630 nm decreased
with increasing concentrations of ONOOÀ; a new emission
band around 480 nm appeared, accompanied by a fluorescence
color change from orange red to cyan (Fig. 2). The intensity
ratio I480/I630 reached a plateau when 11 equiv. ONOOÀ was
added (Fig. S4, ESI†). A linear relationship between the inten-
sity ratio and the concentrations of ONOOÀ was obtained from
0 to 2 mM (Fig. S5, ESI†) and the detection limit of 3a toward
To elucidate the reaction mechanism, a mixture of 3a and
1
ONOOÀ was extracted for MS analysis and H NMR. As depicted
in Fig. S10 and S11 (ESI†), apparent peaks at m/z 362.1057 and m/z
272.0720 were observed, corresponding to 3a-1 ([3a-1 + H]+:
362.1070) and 3a-2 ([3a-2 + H]+: 272.0740), respectively. A typical
1
signal peak at 9.77 ppm in the H NMR spectrum also supported
the formation of the aldehyde product 3a-2 (Fig. S12, ESI†). Accord-
ingly, a successive oxidation process of 3a by ONOOÀ was proposed
in Scheme 1b. The transformation of the sulfur atom to sulfoxide
proceeded, rapidly leading to an immediate intensity decrease in 3a
at 630 nm and a synchronous partial fluorescence enhancement
around 480 nm due to the inhibition of intramolecular charge
transfer. The subsequent oxidative hydrolysis of the CQN bond
of 3a-1 was slower, affording 3a-2 with a stronger fluorescence.
Fig. 1 (a) Emission spectra of 3a (10 mM) before and after the addition of
various reactive species (100 mM) in PBS solution (pH 7.2–7.4, 10 mM,
containing 1 mM Triton X-100). lex = 370 nm, slit width: 6 nm/6 nm.
(b) The intensity ratio changes of 3a before and after the addition of various
À
reactive species. (1) Blank; (2) ClOÀ; (3) H2O2; (4) ONOOÀ; (5) O2
;
ꢀ
t
2À
(6) BuOOH; (7) ꢀOH; (8) S2À; (9) SO3
.
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Fig. 3 Confocal fluorescence images of endogenous ONOOÀ in MCF-7
cells. (top) MCF-7 cells were stained with 10 mM 3a for 30 min. (bottom)
MCF-7 cells were incubated with LPS (1 mg mLÀ1) and IFN-g (50 ng mLÀ1
)
for 24 h and then cultured with 3a for another 30 min. Scale bar: 25 mm.
Cyan channel: lex = 405 nm, lem = 450–510 nm; red channel: lex = 405 nm,
Scheme 1 (a) Structures of probes 3a–3c; (b) the proposed reaction
between 3a and ONOOÀ.
lem = 610–670 nm.
Therefore, a fast intensity enhancement at 480 nm in the first
few seconds with a slower increment in the next few minutes was
observed in the kinetics test (Fig. S9a, ESI†).
5-FU, along with a simultaneous brightness increment in the
cyan channel. The intensity ratio (cyan/red) obviously changed,
suggesting that ONOOÀ was up-regulated in EC1 and MCF-7
cells treated by 5-FU and it might play a vital role in killing
cancer cells. To the best of our knowledge, this is the first
example of visualization of ONOOÀ generation in cancer cells
during chemotherapy, which demonstrated the practicality of
probe 3a.
The desirable fluorescence properties of 3a for ONOOÀ encour-
aged us to extend its utility to intracellular ONOOÀ detection. The
cytotoxicity of 3a in the human breast adenocarcinoma cell line
(MCF-7 cells) was measured first. After incubation with various
concentrations of 3a for 24 h, more than 95% of the cells were
found to be viable when 10 mM of the probe was added. More than
80% of the cells survived even when cultured with 30 mM of the
probe, suggesting the outstanding biocompatibility of probe 3a
(Fig. S13, ESI†).
Moreover, ONOOÀ production in 5-FU treated-normal cells,
human esophageal epithelial Het-1A cells was monitored to
understand its drug safety. Unlike in cancer cells, the intensity
ratio varied little in Het-1A cells incubated with 50 mM of 5-FU,
indicating that the drug might show a weaker cytotoxicity to
normal cells (Fig. S16, ESI†). However, evident changes in ratio
color were visualized upon increment in the amount of 5-FU
(100 and 200 mM), suggesting ONOOÀ up-regulation induced by
the overdose of 5-FU in Het-1A cells. Hence, ONOOÀ can serve
as a biomarker for assessing the safety of 5-FU in normal cells.
Next, the detection of exogenous and endogenous ONOOÀ in
MCF-7 cells using 3a was carried out. When cells were cultured
with 10 mM of probe 3a for 30 min, moderate red fluorescence
and a weak cyan emission were observed. When 100 mM
3-morpholinosydnonimine (SIN-1, a ONOOÀ donor) was added
for incubation for another 30 min, the red fluorescence nearly
disappeared with apparent ratio changes (cyan/red) (Fig. S14, ESI†).
The cells were further incubated with lipopolysaccharide (LPS) and
interferon-g (IFN-g) for 24 h, and then incubated with 3a for
another 30 min. The fluorescence from the red channel diminished
along with the emission enhancement from the cyan channel
yielding an obvious ratio change in the overlay image (Fig. 3).
The combined results showed that 3a is nicely cell-permeable and
can sensitively detect intracellular ONOOÀ in a ratiometric manner.
5-FU is a potent drug for the treatment of a variety of cancers
by inhibiting DNA and RNA synthesis to drive cell death.17
Recently, 5-FU-loaded nanoparticles have been reported to
induce cell apoptosis in an ROS-dependent manner.18 However,
the augmented oxidative stress in cancer cells induced by 5-FU
onꢀly has not been evidenced visually. As ONOOÀ is formed by
O2 À and NO, it is regarded as an indirect indicator of oxidative
stress. Thus, we used our probe to monitor ONOOÀ fluctuation
to indirectly report oxidative stress in 5-FU-stimulated cancer
cells, including MCF-7 cells and esophageal carcinoma cell line
EC1 cells. MCF-7 cells and EC1 cells were treated with various
concentrations of 5-FU (0 mM, 50 mM, 100 mM) for 20 h,
respectively, followed by the culture with probe 3a. As seen in
Fig. 4 and Fig. S15 (ESI†), the emission from the red channel
decreased when both cancer cells were treated with 50 mM of
Fig. 4 Detection of ONOOÀ generation in 5-FU-treated EC1 cells. (a) EC1
cells were stained with different amounts of 5-FU (0 mM, 50 mM, 100 mM)
for 20 h. Then, probe 3a (10 mM) was added for incubation for another
30 min before confocal imaging. (b) Intensity ratio (cyan/red) changes
versus different concentrations of 5-FU. Error bars are Æ SEM. Scale bar:
25 mm. Cyan channel: lex = 405 nm, lem = 450–510 nm; red channel: lex
405 nm, lem = 610–670 nm.
=
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J.-T. H. acknowledges grants from the National Natural Science
Foundation of China (no. 21807029), Key Laboratory of Emer-
gency and Trauma (Hainan Medical University), and Ministry of
Education (Grant KLET-201904); S. W. acknowledges grants from
Nanhu Scholars Program for Young Scholars of XYNU. This
research was also supported by the Analysis & Testing Center of
Xinyang Normal University.
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